PROJECT SUMMARY The goal of this project is to determine if targeting the selectin-ligand, PSGL-1 (P-selectin glycoprotein-1), represents a novel strategy to improve immune-mediated destruction of melanoma potentially by relieving multiple aspects of immunosuppression, a key element that must be achieved for tumor eradication. PSGL-1 is expressed exclusively by hematopoietic cells, and is a highly conserved ligand for the selectin family of adhesion molecules, P, E, and L, that regulate cell migration, but also binds other ligands that include chemokines and extracellular matrix components in both humans and mice. Our studies of chronic viral infections indicate that PSGL-1 is a previously unrecognized negative regulator of T cell responses that can also prevent effective anti-tumor responses to melanoma. In a chronic viral infection model with the LCMV-variant, Clone 13, PSGL-1-deficient mice completely prevent the establishment of chronicity. These mice developed CD8+ and CD4+ effector T cells that were multifunctional and failed to develop the hallmarks of exhaustion characterized by expression of multitude of inhibitory receptors including, PD-1, Lag3, CD160, BTLA and Tim3. PSGL-1-deficient T cells had improved survival leading to maintenance of a significantly greater number of functional effector T cells. Mechanistically, ligation of PSGL-1 on exhausted CD8 T cells inhibited TCR signaling and was linked to upregulation of PD-1. In two melanoma cancer models where T cells are also suppressed, we found that PSGL-1 was highly expressed on all hematopoietic cells in the tumor microenvironment (TME). PSGL-1-deficient mice showed dramatic control of melanoma growth that was coupled to greater numbers of tumor infiltrating, multifunctional CD8+ and CD4+ T cells (TILs) that expressed lower levels of inhibitory receptors. Since targeting PSGL-1 leads to downregulation of multiple inhibitory receptors and promotes effective anti-tumor responses, there may be significant advantages to blocking this receptor compared to combinations of inhibitory receptors, particularly if toxicity is less, or not greater than with current checkpoint blockades. Furthermore, blocking PSGL-1 may have the potential to limit immune suppression in patients that are unresponsive or have suboptimal responses to other therapies. For this project we have generated PSGL-1 conditional KO (PSGL-1fl/fl) mice to interrogate the function of this receptor on lymphoid and myeloid cells in the melanoma TME by cellular and transcriptome analyses. We will use pharmacologic approaches with genetic validation to determine if blocking PSGL-1 function can control melanoma growth by modulating TILs. These studies will provide new insights into the regulation of immune responses by PSGL-1 and mechanisms by which this receptor can contribute to T cell dysfunction in cancer. Most importantly, these studies will establish whether PSGL1 is a new target for immunotherapy of cancer.